The objectives of this project are to determine the biophysical underpinnings of soluble and peripheral membrane protein transport and localization within retinal photoreceptors. Photoreceptors are highly polarized neurons with cellular functions segregated into discrete compartments. Light signaling takes place in the ciliary outer segments, a specialized compartment that contains the phototransduction machinery. The levels of some components of the phototransduction cascade are modulated in a signal-dependent manner through mechanisms that are not understood. Competing biophysical mechanisms appear to be at play, including protein oligomerization, membrane association via post-translational lipidation and steric volume exclusion (SVE). The various biophysical components will be systematically examined using live cell multiphoton/confocal fluorescence imaging. Fluorescent protein probes or fusions of endogenous proteins in retinal photoreceptors will be expressed in transgenic Xenopus laevis or mouse photoreceptors. Examination of protein dynamics and diffusion will be achieved using multiphoton fluorescence recovery after photoconversion (FRAP) using photo-switchable fluorescent proteins and fluorescence correlation spectroscopy (FCS). Protein-protein interactions in live cells will be examined by Forster resonance energy transfer (FRET) and fluorescence cross correlation spectroscopy (FCCS). Additionally protein oligomerization and protein-protein associations, in vitro, will be assessed by small angle X-ray scattering (SAXS) and sedimentation velocity (SV) or sedimentation equilibrium (SE). Protein lipidation states from photoreceptor extracts will be analyzed by mass spectrometry.
Specific aims :
Aim 1. Determine how protein multimerization and DSVE control transport and localization of arrestin-1 in rods.
Aim 2. Determine how transducin membrane association governs its distribution and mobility in rods.
Aim 3. Determine how lipid-shielding chaperone proteins promote solubility and the light-dependent transport of transducin subunits between the major rod compartments.
This work seeks to understand the molecular mechanisms of retinal photoreceptor function in health and disease. Retinal degeneration and blindness may be caused by improper delivery of key proteins to the cells in the eye that detect light. Understanding the mechanisms that control this delivery, and what goes wrong in blinding diseases, will help find new therapies to extend or restore vision.
